An estimated 170 million people worldwide are infected with hepatitis C virus (HCV), including about 4 million in the United States. Approximately 80% of seropositive individuals develop chronic infections predisposing the infected patient to the development of fibrosis, cirrhosis and hepatocellular carcinoma. HCV replication is thought to occur on the surface of rearranged membranes derived from the endoplasmic reticulum(ER), consistent with the schemes utilized by other positive-strand RNA viruses. However, the role of individual HCV proteins and the organization and function of the replication complex has not been defined, and thus the fundamental mechanisms of membrane-associated HCV replication remain unknown. The focus of our studies is non-structural protein 4B (NS4B), an integral ER membrane protein, whose function in HCV replication is the least understood not only in HCV, but also in other members of the Flaviviridae family. Pertinent to this proposal, we have recently shown by exchanging the NS4B coding region between two closely related HCV isolates that allelic differences in NS4B result in replication changes as high as1000- fold. Furthermore, the amino- and carboxy-termini of NS4B harbor sequences essential for efficient replication of the HCV genome. Our long-term goal is to define the role of NS4B in HCV replication and pathogenesis. In this proposal the objectives are to identify the fundamental NS4B sequences required for NS4B function and determine how NS4B participates in HCV replication by defining the steps in the HCV replicative cycle where NS4B acts, and identifying NS4B-mediated interactions with other HCV proteins required for RNA replication. Collectively, these studies will provide valuable information on the role(s) of NS4B in two independent HCV isolates and provide a better understanding of how NS4B contributes to the assembly of a functional replication complex. This data will also advance our understanding of the molecular mechanisms of HCV replication and could suggest potential regions within NS4B or interaction surfaces between NS4B and other HCV-encoded proteins that may be useful for designing anti-viral drugs to combat HCV infection.